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Volume 52 (5): 557-566, 2004
Copyright ©The Histochemical Society, Inc.
Uptake and Rapid Transfer of Fluorescent Ceramide Analogues to Acidosomes (Late Endosomes) in Paramecium
Pacific Biomedical Research Center, University of Hawaii at Manoa, Honolulu, Hawaii
Correspondence to: Dr. Richard D. Allen, Pacific Biomedical Research Center, University of Hawaii at Manoa, 2538 The Mall, Snyder Hall 306, Honolulu, HI 96822. E-mail: allen{at}pbrc.hawaii.edu
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The ciliated protozoan Paramecium incorporates sphingolipids into its cell membranes. However, it is still unclear if these sphingolipids are metabolically synthesized in the cell or if their precursors are taken up from exogenous materials. Here we studied the route of uptake of fluorescence-labeled analogues of ceramide. Fluorescent ceramide was taken up rapidly independent of phagosome formation. Cold treatment caused a decrease in uptake, while reduction in the amount of cytosolic ATP induced by NaN3 and deoxyglucose resulted in accumulation without internalization of fluorescence at the plasma membrane. These results suggest that uptake of fluorescent ceramide occurs at the plasma membrane, that it is an ATP-dependent process, and that it is not a result of simple diffusion. At first intracellular fluorescence appeared principally in the posterior half of the cell and then spread throughout the cytosol. In particular, a high accumulation of fluorescence occurred in association with acidosomes (late endosome or multivesicular body-like vesicles) that bind to the surface of nascent and young phagosomes. Therefore, in the Paramecium cell a significant proportion of ceramide apparently enters the cell by endocytosis and is quickly relayed to acidosomes along the endocytic pathway before becoming part of the digestive vacuole (phagoacidosome) membrane. (J Histochem Cytochem 52:557–565, 2004)
Key Words: ceramide uptake • acidosome • phagocytosis • membrane traffic • fluorescence microscopy • Paramecium
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Introduction |
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SPHINGOLIPIDS AND GLYCERO-PHOSPHOLIPIDS are widely distributed in animal cells and are essential components of plasma membranes. The functions of sphingolipids in cells were at first unclear, but now their physiological roles are being revealed. Sphingomyelin is a major component of lipid microdomains, referred to as lipid rafts (Harder and Simons 1997
), in the plasma membrane. Lipid rafts accumulate cholesterol in addition to sphingomyelin and are also the site for accumulation of certain specific membrane proteins. These proteins are often involved in signal transduction (Simons and Toomre 2000) and the sphingolipids themselves act as second messengers in the signal transduction pathway, e.g., in apoptosis (Ohanian and Ohanian 2001; Cuvillier 2002).
To study the uptake, transport, localization, and metabolism of sphingolipids, fluorescent analogue molecules of sphingolipids have been developed that can be visualized in the living cell (Pagano et al. 2000). For example, in many studies on mammalian cells, fluorescent ceramide added exogenously is taken into the cell and after 30 min, more or less, becomes concentrated in the Golgi apparatus (Lipsky and Pagano 1983). Ceramide is then metabolized to sphingomyelin or glucosylceramide in the Golgi apparatus before it is translocated to the plasma membrane (Pagano et al. 1991).
In protozoa, only a few studies of sphingolipids have been carried out. These studies show biochemically that sphingolipids are present in cell homogenates and especially in the ciliary membranes of Paramecium (Andrews and Nelson 1979; Rhoads and Kaneshiro 1979) and Tetrahymena (Kaya et al. 1984). Furthermore, precise studies were done to classify the subtypes of sphingolipids in P. tetraurelia (Kaneshiro et al. 1984,1997). Glycosyl-phosphatidylinositol (GPI)-anchored proteins, which are known to accumulate in lipid rafts, have also been studied in ciliated protozoa (Capdeville and Benwakrim 1996; Zhang and Thompson 1997; Paquette et al. 2001). These studies suggest that ciliated protozoa, like mammalian cells, have lipid rafts in their plasma membrane. In fact, it was shown that the presence of rafts in the marine ciliate Parauronema (Sul and Erwin 1997) and changes in the sphingolipid composition of ciliary membranes can have a pronounced effect on the activities of Ca2+ and K+ channels that reside in the ciliary membranes of P. tetraurelia (Forte et al. 1981).
Our interest in the lipid composition of membranes of Paramecium has grown out of our previous work, particularly on the contractile vacuole (CV) membrane. The CV rounds up just before fluid is discharged from the cell but this rounding does not appear to be caused by a contractile actomyosin system. Rather, it appears to be an inherent property of the membrane itself. When the cell is disrupted and the CV is released from the cell but is still bathed in cytosolic fluid, the CV can be seen to periodically round up and relax even though it is no longer able to fuse with the plasma membrane to release its fluid content (Tani et al. 2000). In fact, any membrane fragment derived from the smooth spongiome, from which the CV membrane is also derived, is capable of a similar in vitro cycle of rounding and relaxing activity (Tominaga et al. 1998; Tani et al. 2000), which can continue in the cell-disrupted state for as long as 30 min (Tani et al. 2001). The cyclic activity appears to be under the control of a timing mechanism that is built into the membrane itself. During this rounding, the tension of the membrane increases 35-fold over its tension in the relaxed state (Tani et al. 2001). We have proposed that tension development is under the control of changes in spontaneous curvature of the CV membrane (Tani et al. 2002).
To study the biochemical properties of this very dynamic membrane we have exposed the cell to fluorescent ceramide analogues to see in which endomembrane systems of Paramecium this lipid will accumulate. Membranes that show an ability to curve into pits and tubules often contain sphingolipids and cholesterol and, in some cases, the enzyme sphingomyelinase or its regulatory proteins are associated with membranes involved in such curvature (Zha et al. 1998; Gerald et al. 2002). Here we report that Paramecium cells quickly take up fluorescent ceramide at the plasma membrane and that this can occur even in the absence of phagocytosis. This process required cytosolic ATP for energy, even though ceramide, which is an amphipathic molecule, can presumably diffuse directly into the plasma membrane in mammalian cells (Putz and Schwarzmann 1995). A fluorescent ceramide analogue in Paramecium cells soon appeared in the cytosol, where it was quickly transferred to acidosomes rather than to the Golgi apparatus as has been reported for mammalian cells. We also observe some labeling of the contractile vacuole complex.
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Materials and Methods |
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Cells
Paramecium multimicronucleatum (syngen 2) was cultured in an axenic medium as previously described (Fok and Allen 1979
). Cells in the mid-logarithmic growth phase were centrifuged (
120 x g) for 25 sec to form a loose pellet. The cells were then suspended in a saline solution (2 mmol l–1 KCl, 0.25 mmol l–1 CaCl2, 80 mmol l–1 sorbitol, 1 mmol l–1 MOPS–KOH, pH 7.0) containing 0.2% (w/v) bovine serum albumin (BSA). This solution has an osmolarity of 84 mosmol l–1, which is almost identical to that of the axenic culture medium. Cells were washed twice with this saline solution. Cell culture and experimentation were performed at a room temperature of 24 ± 1C, unless otherwise noted, which was regulated by a window-type air conditioner.
Treatment of Cells with Fluorescent Ceramide Analogues
Cells were incubated in a saline solution containing the fluorescent ceramide (Cer), which was either 6-((N-(7-nitrobenz-2-oxa-1,3-diazo-4-yl)amino)hexanoyl)-sphingosine (NBD C6-Cer) or 0-N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine (BODIPY FL C5-Cer) at concentrations of 0-30 µmol l–1. NBD-Cer was used mainly when double staining experiments were required because BODIPY FL fluorophore emits a red fluorescence when it is highly concentrated at a wavelength of 620 nm, which is near the emission of AlexaFluor 568 fluorophore (603 nm), which we used as secondary antibody in the indirect-immunofluorescence procedure. The fluorescent Cer analogues were purchased from Molecular Probes (Eugene, OR). Stock solutions of the fluorescent Cer were prepared as 5 mmol l–1 solutions dissolved in dimethylsulfoxide (DMSO). An aliquot of living cell suspension containing fluorescent Cer was put on a slide and pressed under a coverslip to retard cell movement for microscopic observation. To prevent phagosome formation, 300 µmol l–1 of cytochalasin B (CB) (Sigma, St Louis, MO; dissolved in DMSO at 150 mmol l–1 as a stock solution) was added to the cell suspension.
Cold Treatment and ATP Depression
In one experiment, after a 30-min precooling period at 4C, cells, kept at 4C, were incubated for 20 min with BODIPY-Cer. In a second experiment, ATP synthesis was inhibited by the addition of 5 mmol l–1 NaN3 and 50 mmol l–1 2-deoxyglucose (both from Sigma). This mixture of inhibitors was prepared in a saline solution at a concentration that kept the final osmolarity of the solution at 84 mosmol l–1 by modifying the concentration of sorbitol. The inhibitors were present in the cell suspension for the entire 20-min incubation in BODIPY-Cer and during its 30-min pretreatment. The concentration of BODIPY-Cer in the cell suspension was 15 µmol l–1. To prevent phagosome formation, 300 µmol l–1 of CB was added at 5 min before the addition of BODIPY-Cer in all cases, including a control experiment.
Pulsing Phagosomes with Latex Beads
Cells were initially incubated with 15 µmol l–1 BODIPY-Cer for 30 min and then latex beads of 0.8 µm in diameter were added to the cell suspension for various times at a final bead concentration of 0.004% (w/v). The cells were incubated continuously until they were fixed for 20 min with 3% (w/v) formaldehyde in 50 mmol l–1 phosphate buffer (pH 7.4). After fixation the cells were washed once for 20 min with PBS before observation.
Immunostaining with Anti-vacuole Monoclonal Antibodies
After incubation of living cells with 15 µmol l–1 NBD-Cer for 60 min, cells were washed twice with saline solution, spun down in a centrifuge after each wash, and fixed with formaldehyde for 30 min. Fixed cells were permeablized with cold (–20C) acetone for 20 min. Treatments with primary monoclonal antibodies (MAbs) to digestive vacuoles, which were anti-DV-I, anti-DV-II, and anti-DVIII MAbs, were for 60 min each and that with the secondary antibody, which was AlexaFluor 568-goat anti-mouse IgG (Molecular Probes), was for 30 min. Two 20-min washes with PBS were carried out between each step.
Microscopic Observation and Fluorescence Intensity Measurements in Cells
Observation of fluorescent cells was carried out using a fluorescent microscope (Eclipse E400; Nikon, Tokyo, Japan) equipped with epifluorescence illumination and appropriate filters (Nikon), which were B-2E used for BODIPY FL and NBD, and Y-2E for AlexaFluor 568. Photographs of fluorescent images were taken with a digital camera (Coolpix 4500; Nikon).
Fixed cells were used for measurements of the total amount of fluorescence in a cell body. After incubation of living cells with 15 µmol l–1 BODIPY-Cer for a particular period, an aliquot (0.75 ml) of cell suspension was diluted to total 15 ml with saline solution and centrifuged to allow supernatant removal. Cells were then fixed 30 min with 15 ml of formaldehyde solution in which BSA was deleted. The concentration of BODIPY-Cer remaining in the fixative solution was estimated to be less than 0.005 µmol l–1. After fixation the cells were washed twice with BSA-free PBS for 20 min and then mounted on a slide with 2.5% (w/v) 1,4-diazobicyclo-[2,2,2]-octane dissolved in a mixture of 30% (v/v) phosphate buffer and 70% (v/v) glycerol to retard photobleaching of the fluorescence (Allen et al. 1988). The integrated fluorescence intensity of each cell was measured from the digital photo image using NIH image 1.62 software (downloaded from http://rsb.info.nih.gov/nih-image/). The linearity of the measurement corresponding to the actual fluorescence intensity under our experimental conditions was verified by preliminary experiments that measured the fluorescence of objects excited by different intensities of UV through the ND filters.
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Uptake and Intracellular Localization of Fluorescent Ceramides
To follow the course of Cer entry into the Paramecium cell, living cells treated with 15 µmol l–1 BODIPY-Cer were observed microscopically for fluorescence localization over a period of time of up to 40 min. As shown in Figure 1A , the fluorescence was present in the cytoplasm in both the CB-treated cells and in the untreated (control) cells after 5 min in BODIPY-Cer. In control cells, fluorescence appeared concentrated at first in the posterior half of the cytoplasm, particularly around a few phagosomes. As early as 5 min, one vacuole could also be seen to contain fluorescence only in its lumen in the posterior part of the cell. In addition, many vesicles (
0.5 µm in diameter) with increased levels of fluorescence were seen to border some phagosomes in the posterior part of 5–10-min-incubated control cells (Figure 2A)
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By 40 min, fluorescence had spread throughout the cytoplasm, while macronuclei were less stained in both control and CB-treated cells. A wide rim of fluorescence was seen around a few vacuoles (asterisks in Figure 1A), and a few vacuoles with a high concentration of fluorescence in their lumens were still observed in control cells. Even though the CB-treated cells after 40 min for the most part lacked phagosomes, strong fluorescence could often be observed at the cytopharynx (arrowhead in Figure 1A). Previous studies have shown that cells can sometimes form a phagosome even when the CB is present, and these vacuoles can be very large (Fok et al. 1985
). In such cells, fluorescence tended to accumulate at the vacuole membrane, or in vesicles surrounding these vacuoles, to a greater extent than in the control cells (asterisk in Figure 2B).
When cells that had been incubated with fluorescent Cer for 60 min were fixed and the excess intracellular Cer analogue was absorbed by 0.2% BSA in PBS (back-exchange procedure; Pagano et al. 1989), fluorescence was also found in the contractile vacuole complex (CVC) (Figure 2C). The CVCs were also observed to be stained even in cells that had been incubated with Cer analogue for only a few minutes, although the fluorescence was very weak (data not shown). The CVC was not recognizable in living cells because the background cytosolic fluorescence was apparently too bright for the CVC to be visible.
The total amount of BODIPY-Cer in cells increased rapidly during the first 15 min and then more gradually during the next 15–20 min (Figure 1B). The initial uptake of Cer within the first 5 min in control cells was linear and was identical to that in CB-treated cells. However, beyond this time fluorescence entered CB-treated cells at a reduced rate compared with control cells. This increased amount of fluorescence in the control cells might be caused by those vacuoles that have a strong fluorescence in their lumens that are not present in CB-treated cells. The initial rate of uptake during the first 5 min depended directly on the external concentration of BODIPY-Cer (Figure 1C). Similar observations were made when NBD-Cer was used instead of BODIPY-Cer, except for an overall weaker fluorescent emission than was attainable with BODIPY-Cer.
Effects of Cold Treatment or ATP Depression on Uptake of Ceramide
To determine whether or not the uptake of Cer in Paramecium cells is a simple diffusion, we followed Cer uptake in cold-treated cells and when an inhibitor of ATP synthesis was applied. Compared with control cells (Figure 3A)
, uptake of Cer was suppressed dramatically at 4C (Figures 3B and 3D) (p<0.0001, t-test). Inhibiting ATP synthesis by adding NaN3/deoxyglucose did not reduce the total amount of Cer fluorescence in the cells. In fact, fluorescence increased significantly over the control cells (Figure 3D) (p<0.0001). However, the fluorescence was apparently localized at the plasma membrane (Figure 3C), excluding ciliary membranes, whereas it was present throughout the cytoplasm in control cells (Figure 3A). These results suggest that the fluorescent Cer analogue does not diffuse across the plasma membrane in cold-treated cells or when ATP synthesis is inhibited and ATP is depleted.
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Stage and Age of Vacuoles That Exhibit BODIPY Fluorescence at Their Surface or in Their Lumens
To determine the age of phagosomes with which Cer is associated, cells were pulsed with latex beads for different periods of time. As shown in Figure 4 , fluorescence had already accumulated in a 2-min-old vacuole. However, vacuoles younger than 2 min were unstained. In the 6-min-pulsed cell in Figure 4, only one vacuole of six that contained latex beads was associated with fluorescence. The fluorescence of BODIPY-Cer in fixed cells diffused away from the area around the phagosomes and became dim. Latex beads may also obscure the fluorescence inside the vacuole.
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Because vacuoles that had strongly fluorescent materials in their lumens were not co-labeled with latex beads after short pulsation periods (
10 min), we used stage-specific MAbs against different digestive vacuole stages to determine the relative age of the vacuoles that did contain strong fluorescence. After a 40-min incubation with NBD-Cer, cells were fixed and treated with a group of vacuole-specific MAbs. The vacuoles containing NBD-fluorescent materials were stained with anti-DV-III MAb (Figure 5) but not with anti-DV-I or -DV-II MAbs when these two MAbs were used. This means that the vacuoles containing strong luminal fluorescence were in a late stage of the digestive cycle. In accord with this observation, as illustrated in Figure 5, many of the vacuoles with strong luminal fluorescence were found in the posterior region of the cell near the cytoproct. |
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Ceramide Analogue Uptake at the Plasma Membrane Is ATP-dependent in Paramecium
Fluorescent Cer analogue enters human fibroblasts quickly, even at 2C, and when the temperature is raised to 37C it accumulates in the Golgi apparatus (Lipsky and Pagano 1985
). Therefore, in this cell system uptake appears to occur by an ATP-independent simple diffusion, even though the authors considered the possibility of an unknown facilitating process. However, the uptake of fluorescent Cer in Paramecium did not appear to be a simple diffusion across the plasma membrane because cold treatment did reduce the rate of uptake and the reduction of ATP in the cell limited the accumulation of Cer to the plasma membrane or, at least, to the cell's pellicle (Figure 3C). Because uptake of Cer within the first 10 min occurs basically across the plasma membrane or via endocytosis, entry via phagosome formation can be ruled out. Our results suggest that fluorescent Cer is initially integrated into the plasma membrane, which is dependent on temperature and not on ATP, and then is internalized by an ATP-dependent process. In addition, because the rate of the initial uptake is absolutely dose-dependent (Figure 1C), the integration of Cer analogue into the plasma membrane is not a saturable Cer-selective process such as would be expected if receptors for Cer were involved.
Paramecium cells have an endocytic pathway similar to that of other cell types (Allen and Fok 2000). Because the uptake of Cer analogue into the cytosol is an energy-dependent process, endocytosis may be involved. Classical receptor-mediated endocytosis requires ATP for clathrin cage disassembly, while the assembly process requires GTP. In Amoeba proteus, Topf et al. (1996) proposed the existence of a transporter molecule for Cer uptake, such as a "flippase" (Devaux 1992), to transport Cer across the plasma membrane's lipid bilayer. Like Paramecium, this protozoan takes up Cer analogue by an ATP-dependent process that may or may not follow the endocytic pathway. In any event, plasma membranes of protozoa appear to be less permeable to Cer analogues than are mammalian cells, and an ATP-dependent transport system is needed for Cer to cross the plasma membrane and to enter the cytoplasm.
Intracellular Transport and Localization of the Internalized Ceramide Analogue
In the cytoplasm of living Paramecium, fluorescent Cer had already accumulated around digestive vacuoles by 5 min (Figure 1A). This dramatic and rapid localization of fluorescence resulted from the presumed accumulation of fluorescence in the membranes of many vesicles that were 0.5 µm in diameter and that are known to form a layer around one or two digestive vacuoles (Figure 2A). On the basis of pulse-chase experiments, using latex beads as the pulse, it is clear that these digestive vacuoles, surrounded by the fluorescence-associated vesicles, were young phagosomes 2 min old or less (Figure 4). The vesicles that accumulate around such young phagosomes in the posterior of the cell have been defined as acidosomes (Allen and Fok 1983c; Allen et al. 1993).
That these vesicles are acidosomes is also supported by an experiment in which digestive vacuole formation is suppressed by the addition of CB. In this experiment, fluorescence was concentrated around a huge digestive vacuole (Figure 2B) that is typical of CB-treated cells (Allen and Fok 1983a). This phenomenon is the result of a layer of acidosomes surrounding the nascent phagosomal membrane. The acidosomes bind only to the cytopharynx and nascent vacuole membrane and to one or two early phagosomes. The pinching off of phagosomes and subsequent fusion of these acidosomes with these early phagosomes is blocked by CB treatment (Allen and Fok 1983b). In Figure 1A (arrowhead), accumulation of fluorescence was observed at the nascent vacuole. Because acidosomes also line up along the cytopharyngeal microtubular ribbons (mixed together with the discoidal vesicles and with carrier vesicles) (Allen and Fok 2000), acidosomes might be expected to be found in the oral region even though an expanded nascent vacuole is absent at the cytopharynx.
What route does fluorescent Cer take from the plasma membrane to the acidosomes? Because specific accumulation of fluorescence into acidosomes occurred rapidly even when cytosolic fluorescence was still weak, at least some of the transfer of fluorescent Cer seems to have followed the endosomal pathway. Allen et al. (1993) reported that clathrin-coated vesicles pinching off the plasma membrane at the parasomal sacs rapidly fuse with early endosomes after the vesicles lose their coats. Carrier vesicles (100 nm) are then budded from the early endosomes and these are transported to acidosomes along the cytopharyngeal microtubular ribbons, where they fuse with the acidosomes. Therefore, Cer diffusing into the plasma membrane can enter the endocytic pathway at the coated pits and, in time, the Cer will be transferred to the acidosomes as part of the trafficking membrane of the endocytic pathway. The small size of the many compartments of the early endocytic pathway prevents their detection at the light microscopic level. Whether Cer passes into the cell's cytoplasm exclusively through the endocytic pathway or whether some Cer also crosses the plasma membrane by energy-utilizing processes, such as a process utilizing flippases and transport proteins, as is known to be used in phospholipid transport, cannot be determined with fluorescence microscopy alone.
Significance of the Accumulation of Ceramide Analogue in the Acidosomes and CVC of Paramecium
In any event, Cer does rapidly accumulate in acidosomes in Paramecium, which represent the late endosomes in this cell. Acidosomes have characteristics similar to those of multivesicular bodies (MVBs) of mammalian cells. Both the acidosomes and MVBs are regarded as late endosomes because they fuse with vesicles originating from early endosomes (Allen et al. 1993; Shih et al. 2002). In addition, like MVBs, acidosomes show significant membrane invaginations into their lumens (Allen et al. 1993) that resemble the internal membrane structures and vesicles (exosomes) of MVBs (Denzer et al. 2000). However, in the acidosomes of Paramecium the indentations are not known to pinch off to form vesicles. The MVB has recently been found to contain high concentrations of cholesterol in human B-lymphocytes (Möbius et al. 2003). Cholesterol is particularly abundant in the internal membrane material (exosomes) of MVBs. The latter authors speculated that this cholesterol distribution is important for the membrane curvature that leads to vesicular and tubular compartment formation in the MVB. Because cholesterol molecules have smaller overall size and a smaller hydrophilic head group than those of phosphoglycerolipids, the presence of cholesterol in one leaflet of a lipid bilayer might be enough to generate high curvature in the membrane.
Ceramide is similar to the cholesterol molecule in having a small hydrophilic head. Holopainen et al. (2000) showed that, during enzymatic degradation of sphingomyelin to ceramide, a liposome membrane is caused to invaginate and vesiculate. Even exogenous ceramide could induce the change in curvature in the membrane into which it was integrated. Therefore, we speculate that the presence of ceramide in the acidosome membrane may be easily accommodated in a membrane known to have the ability to form tightly curved necks (40 nm) at membrane invaginations. Such necks have been visualized in freeze-fracture images of acidosomes (phagosome fusion vesicles) (see Figure 9 in Allen and Fok 1983b).
The localization of fluorescent Cer was also observed in the contractile vacuole complex (CVC), even though the fluorescence intensity in the CVC was lower than that of acidosomes (Figure 2C). The CVC is formed by highly tubular membranes that have been classified as the smooth and the decorated spongiomes (Allen and Fok 1988; Allen and Naitoh 2002). We anticipate, again, that the CVC membranes, which display high curvature, might have an affinity for Cer analogues and for sphingolipids and cholesterol-like lipids.
Destiny of Ceramide Analogues After Accumulation in Acidosomes
The number of digestive vacuoles with strong fluorescence in or around their membrane usually ranges from zero to three per cell. Therefore, it is believed that fluorescence does not stay in a vacuole membrane for a very long time after fusion of the acidosomes with the young phagosome (DV-I) has occurred. The DV-I vacuole becomes a phagoacidosome (DV-II) by the retrieval of the DV-I membrane and the addition of acidosomes, and soon after this the DV-II fuses with lysosomes to form the phagolysosome (DV-III). The Cer analogue might be removed from the phagosome membrane after fusion with the acidosomes is completed because membrane tubulation, evident when a DV-I becomes a DV-II, is dramatically reduced in the relatively planer DV-II membrane. On the other hand, the Cer analogue may be removed in the phagolysosomes by a lysosomal enzyme such as ceramidase (Chen et al. 1981) as a result of a change in the affinity of the Cer analogue for the DV-III membrane. However, ceramidase has yet to be reported in Paramecium.
What is the origin of the vacuoles that have a very strong fluorescence in their lumens rather than in their membranes, such as those seen in many control cells (Figure 1A)? Such vacuoles were apparently in a late stage of the digestive vacuole cycle because they were never labeled by latex beads in the 10-min pulsation studies (data not shown) and, in addition, their membranes were labeled only by the anti-DV-III MAb (Figure 5) and not by anti-DV-I or anti-DV-II MAbs. Fluorescent material remained in the lumens of these vacuoles even after the procedure for demonstrating membrane immunofluorescence, which included acetone permeabilization, was completed, whereas the fluorescence surrounding young phagosomes diffused away after such treatments. Therefore, it appears that the fluorescence in the lumen of late digestive vacuoles must not be Cer analogues but may be metabolic waste material originating from Cer analogue dumped there by the cell.
We therefore conclude that ceramide analogues pass into the Paramecium cell across the plasma membrane in part by endocytosis. This pathway leads to a rapid accumulation of fluorescent label in the acidosomes, large vesicles that resemble multivesicular bodies whose membranes often invaginate into their lumens. Fluorescence also appears in membranes of the contractile vacuole complex. Therefore, this analogue seems to prefer a membrane environment that is capable of undergoing pronounced bending. The significance of these observations to the rounding cycles of the CV membrane or to membrane tension development, if any, remains to be defined, although the presence of ceramide might promote spontaneous curvature in these membranes.
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Acknowledgments |
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Supported in part by NSF grant MCB 0136362.
We thank Drs Yutaka Naitoh and Kazuyuki Sugino for profitable discussions. We also thank Marilynn S. Aihara for technical support.
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Footnotes |
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Received for publication January 16, 2004; accepted January 21, 2004
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